Cancer immunotherapies based on blockade of immune checkpoints have achieved significant success. However, more than 50% of patients with advanced cancer are not sensitive to this type of immunotherapy. Although the factors that are responsible for cancer resistance are not fully understood, lack of pre-existing T cell infiltration in the tumor microenvironment (TME) is considered to be the most important factor for anti-PD-1 resistance. Additionally, although not absolute, lack of expression of PD-L1 in tumors has been considered to be another important factor. Finally, although not well-established in human cancer, accumulation of regulatory T cells (Tregs) in the TME has been shown to contribute to anti-PD-1 resistance in mouse models. Tregs are known to be expanded in cancer patients and enriched in cancer lesions. In anti-CTLA-4 antibody-treated cancer patients, Treg-depletion in tumor lesion correlated with therapeutic response. Anti-CTLA-4 treatment also significantly enhanced response rate to anti-PD-1 therapy, but with the price of increased grade 3 and 4 toxicity. Thus, developing novel strategies that can overcome these limitations is critical to enhance the efficacy of current cancer immunotherapies. The anti-tumor activity of IL-27 has been appreciated for more than 10 years. However, developing IL-27 into a therapeutic to treat established cancer has not been well achieved. Recombinant adeno-associated viral vectors (rAAV) are highly versatile gene delivery agents for gene therapy. The lack of immunogenicity and toxicity make rAAV arguably the gene therapy vector of choice for human clinical trials. Recently, we have produced IL-27-expressing rAAV (AAV-IL-27) that can efficiently produce IL-27 in recipient mice and made the following novel observations. First, AAV-IL-27 significantly inhibited the growth of a broad-spectrum of tumor types in mice. Second, AAV-IL-27 treatment resulted in dramatic reduction of Tregs without causing autoimmunity. Third, we have found that AAV-IL-27 therapy show strong synergy with PD-1 antibody in inhibiting tumor growth. Based on these observations, we hypothesize that AAV-IL-27 therapy can promote tumor immunity while inhibit autoimmunity, and has a potential to be used alone for cancer therapy or to enhance current immunotherapies. To test this hypothesis, we will first determine how AAV-IL-27 enhances tumor specific T cell responses in the TME. We will then investigate the mechanisms of AAV-IL-27-mediated depletion of Tregs and determine how it enhances tumor immunity without causing autoimmunity. Additionally, we will investigate the potential of the combination of AAV-delivered IL-27 and anti-PD-1 therapy in cancer therapy, determine the potential mechanisms of synergy and evaluate potential autoimmune side effects in the combination therapy. Finally, we will investigate if IL-27-induced T cell phenotypes can be reproduced in human T cells in vivo. The proposed studies will not only reveal new insights of the anti-tumor activity of IL-27, but also lead to a new candidate therapeutic that can work alone or synergistically with anti-PD-1 antibodies for cancer treatment.